Biologically active amides

ABSTRACT

A polypeptide, designated Pancreatic Hormone III, together with pharmaceutically acceptable salts or metal complexes thereof; pharmaceutical formulations of the said polypeptide or its salts or complexes and the preparation of such formulations; and the use of the said polypeptide, salts and complexes in human and veterinary medicine in effecting a reduction in weight or in appetite or in the treatment of obesity, hyperglycaemia or hyperinsulinaemia.

PRIOR APPLICATION

This application is a continuation-in-part of Ser. No. 676,645 filedApr. 14, 1976.

This invention relates to biologically active polypeptides isolatablefrom the pancreas of vertebrate species, to pharmaceutical formulationscontaining these polypeptides and the preparation of such formulations,and to the use of the polypeptides in human and veterinary medicine.

It is known from the literature (for example, Endocrinology 83, 1323(1968) and 93, 558 (1973), and J. Biol. Chem., 250/24, 9369 (1974)) thatby means of acid-alcohol extraction procedures a polypeptide can beisolated from the pancreas of a number of avian and mammalian specieswhich is structurally and pharmacologically distinguishable from theknown pancreatic hormones insulin, somatostatin and glucagon.Immunological and other studies have further indicated that thepolypeptide varies in structure with the species of origin.

The amino acid sequences of the bovine pancreatic polypeptide (BPP) andavian pancreatic polypeptide (APP, from chicken) to which inter aliathis invention relates are set out in Gastroenterology, 47/4, 737(1974). The polypeptides are identified as straight-chain sequences ofthirty-six amino acid residues having identities at fifteen positions.Similar polypeptides have been isolated from the pancreas of pig (PPP),sheep (OPP) and man (HPP) and are taught (Gastroenterology, loc. cit.)as differing structurally from BPP in only one or two residues atpositions 2,6 or 23. (All references recited herein are incorporatedherein by reference thereto).

Of these pancreatic polypeptides APP and BPP in particular have beenextensively investigated as regards biological activity and as recentlyas 1974 it was stated (Gastroenterology, loc. cit.) that thephysiological role of BPP and APP was unknown.

It has now unexpectedly been found by the Applicants that thesevertebrate pancreatic polypeptides (hereinafter collectively referred toas VPPs, which term should be understood to include such polypeptides ofavian, amphibian, piscian, reptilian or mammalian origin) are as a classeffective in lowering the concentration in the blood of both glucose andinsulin when administered to a strain (NZO) of genetically obese,hyperglycaemic and hyperinsulinaemic mice. At the same time thesetreated mice lose weight as compared with untreated control animals andgive normalized values in the oral Glucose Tolerance Test standard inthe art. It has also been found that these VPPs are as a class effectivein reducing the food consumption of obese rats of the Zucker strain ascompared with obese untreated control animals and that when comparedwith such controls the obese VPP-treated animals lose weight.

In consequence of their newly-found activities as above described theVPPs may be used in the treatment of mammals in the fields of both humanand veterinary medicine for the amelioration of a conditioncharacterised by one of more of obesity, hyperglycaemia andhyperinsulinaemia. Thus specific utilities for the VPPs includetreatment of the following conditions:

(a) obesity associated with normal blood levels of both glucose andinsulin;

(b) obesity associated with hyperinsulinaemia but normal blood glucoselevels;

(c) obesity associated with both hyperinsulinaemia and hyperglycaemia,as for example in lateonset (middle-aged) diabetes in man; and

(d) the need or desire to reduce the appetite for food or induce loss ofweight.

In the veterinary field a particularly important application is in thetreatment of domestic pet animals, particularly cats and dogs.

For each of these utilities the amount required of VPP and the frequencyof its administration will vary with the identity of the VPP concernedand with the nature and severity of the condition being treated and isof course ultimately at the discretion of the physician or veterinarian.In general however a suitable dose of VPP will lie in the range of 0.01to 100 μg per kilogram mammal body weight being treated, preferably inthe range of 0.02 to 20 μg per kilogram and most preferably in the range0.02 to 4.0 μg per kilogram. Administration by the parenteral route(intravenously, intradermally, intramuscularly or subcutaneously) ispreferred.

While it is possible for the VPPs to be administered as the rawsubstances it is preferable, in view of their potency, to present themas a pharmaceutical formulation. The formulations, both veterinary andfor human use, of the present invention comprise a VIP together with oneor more acceptable carriers therefor and optionally other therapeuticingredients. The carrier(s) must be "acceptable" in the sence of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof. Desirably the formulations shouldnot include oxidising agents and other substances with which peptidesare known to be incompatible. The formulations may conveniently bepresented in unit dosage form and may be prepared by any of the methodswell known in the art of pharmacy. All methods include the step ofbringing into association the VPP with the carrier which constitutes oneor more accessory ingredients. In general the formulations are preparedby uniformly and intimately bringing into association the VPP with thecarrier(s) and then, if necessary, dividing the product into unitdosages thereof.

Formulations suitable for parenteral administration convenientlycomprise sterile aqueous preparations of the VPPs which are preferablyisotonic with the blood of the recipient. Such formulations may beconveniently prepared by admixing solid VPP with water to produce asolution or suspension which is filled into a sterile container andsealed against bacterial contamination. Preferably sterile materials areused under aseptic manufacturing conditions to avoid the need forterminal sterilisation.

Such formulations may optionally contain one or more additionalingredients among which may be mentioned preservatives, such as methylhydroxybenzoate, chlorocresol, metacresol, phenol and benzalkoniumchloride. Such materials are of especial value when the formulations arepresented in multi-dose containers.

Buffers may also be included to provide a suitable pH value for theformulation and suitable materials include sodium phosphate and acetate.Sodium chloride or glycerin may be used to render a formulation isotonicwith the blood. If desired the formulations may be filled into thecontainers under an inert atmosphere such as nitrogen or may contain anantioxidant, and are conveniently presented in unit dose or multidoseform, for example in a sealed ampoule.

Where a formulation is presented for human or for veterinary use, theneach dosage unit thereof conveniently contains the VPP in an amount inthe range of 0.5 μg to 5.0 mg, preferably 1.0 μg to 1.0 mg and morepreferably 1.0 μg to 200 μg.

It should be understood that excluded from the scope of the presentinvention are non-sterile mixtures which are mere solutions orsuspensions of the known VPPs in solvents and liquids known in theliterature for use in their synthesis and/or isolation by the methodsdescribed therein. Included within the scope of the present inventionare such solutions and suspensions of the known substances which arepharmaceutically acceptable to the intended recipient thereof and whichcontain in addition at least one other pharmaceutically acceptablesubstance.

It will be appreciated that while VPPs form acid addition salts andcarboxy acid salts the biological activity thereof will reside in thebase/acid (polypeptide) therein. These salts may be used in human and inveterinary medicine and presented as pharmaceutical formulations in themanner and in the amounts (calculated as the base) describedhereinabove, and it is then preferable that the acid moiety bepharmacologically and pharmaceutically acceptable to the recipient.Examples of such suitable acids include (a) mineral acids: hydrochloric,hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; (b)organic acids: tartaric, acetic, citric, malic, lactic, fumaric,benzoic, glycollic, gluconic, gulonic, succinic and aryl-sulphonic, forexample p-toluenesulphonic acids.

The pharmaceutically and pharmacologically acceptable salts togetherwith those salts which are not so acceptable (for example salts ofhydrofluoric and perchloric acids) have utility in isolation andpurification of the VPPs, and of course the unacceptable salts are alsovaluable in the preparation of the acceptable salts by techniques wellknown in the art. Those VPPs containing a plurality of free amino groupsmay be obtained in the form of mono- or poly- acid addition salts, or asmixed salts of a plurality of acids.

Those VPPs, such as APP (vid. sup.), which include a histidyl radicalform complexes with pharmaceutically acceptable metals such as zinc, andsuch complexes have been found to exhibit a prolonged period of actionupon parenteral administration as compared with the un-complexedpolypeptides and their acid addition salts. Such complexes may beprepared by techniques analogous to those well known in the art inrespect of insulin and may be used in human and in veterinary medicine,and presented as pharmaceutical formulations, in the manner and in theamounts (calculated as the polypeptide) described hereinabove. Theformation of zinc-insulin complexes is taught in, for example, ActaChemica Scandinavica, 10 (1956) 1455 and 1459, and 11 (1957) 291, 299,439, 484 and 1248.

In the Journal of Biological Chemistry, 250/24, p.p. 9369-9376 (1975),Kimmel, J. R. et al., suggest that the name "pancreatic hormone III" orthe abbreviated version "PH-III" may be used to refer to thepolypeptides referred to herein, and assign to BPP and APP the followingstructures:

    __________________________________________________________________________    BPP                                                                              Ala.Pro.Leu.Glu.Pro.Gln.Tyr.Pro.Gly.Asp.Asp.Ala.                           APP                                                                              Gly.Pro.Ser.Gln.Pro.Thr.Tyr.Pro.Gly.Asp.Asp.Ala.                           1             6       12                                                      BPP                                                                              Thr.Pro.Glu.Gln.Met.Ala.Gln.Tyr.Ala.Ala.Glu.Leu.                           App                                                                              Pro.Val.Glu.Asp.Leu.Ile.Arg.Phe.Tyr.Asp.Asn.Leu.                           13            18      24                                                      BPP                                                                              Arg.Arg.Tyr.Ile.Asn.Met.Leu.Thr.Arg.Pro.Arg.TyrNH.sub.2                    APP                                                                              Gln.Gln.Tyr.Leu.Asn.Val.Val.Thr.Arg.His.Arg.TyrNH.sub.2                    25            30      36                                                      __________________________________________________________________________

Published United Kingdom Patent Specification No. 1,373,310, U.S. Pat.No. 3,842,063 and Swiss Pat. No. 551, 949 (each of which is incorporatedherein by reference hereto) teach inter alia structures for VPP's(Pancreatic Hormone III) of respectively bovine, porcine, ovine andhuman origin, together with the extraction and isolation of thepolypeptides from pancreas and their characterization. VPP's of knownstructure may be synthesised by classical peptide synthetic methods andVPP's of other species may be isolated from pancreas by proceduresanalogous to those taught in the patent specifications and otherliterature incorporated herein by reference. Such procedures arethemselves analogous to classical extraction procedures used for theseparation of insulin and typically include an initial acid/alcoholextraction of the pancreatic material, removal of lipid, concentration,and fractional separation of the insulin, glucagon and VPP (PancreaticHormone III) by one of more of a variety of methods including gelfiltration, ion exchange chromatography, electrophoresis andcountercurrent distribution.

It will be appreciated from the foregoing that what we will claim maycomprise any novel feature described herein, principally and notexclusively, for example:

(a) A pharmaceutical formulation which comprises a polypeptidedesignated Pancreatic Hormone III or a pharmaceutically acceptable saltor metal complex of said polypeptide, together with a pharmaceuticallyacceptable carrier therefor.

(b) A sterile parenterally acceptable pharmaceutical formulation whichcomprises a polypeptide, or a salt or complex thereof as defined in (a)above together with a pharmaceutically acceptable carrier therefor.

(c) A formulation as defined in (a) or (b) above wherein the polypeptideis isolatable from a mammalian species, for example ox, sheep or pig.

(d) A method of preparing a pharmaceutical formulation as defined in(a), (b) or (c) above which comprises admixture of the polypeptide, or asalt or complex thereof with the carrier therefor and, whereappropriate, sterilization and/or division of the product into unitdosages thereof.

(e) A method for treating a mammal for a condition selected fromobesity, hyperglycaemia and hyperinsulinaemia which comprises theadministration to the mammal of a non-toxic, obesity reducing,hyperglycaemic or hyperinsulinaemic treatment effective amountrespectively of a polypeptide, or a salt or complex thereof as definedin (a) or (c) above.

(f) A method of reducing the weight or reducing the appetite of a mammalcomprising the administration to the mammal of a non-toxicweight-reducing or appetite-reducing treatment effective amountrespectively of a polypeptide, or a salt or complex thereof as definedin (a) or (c) above.

(g) A method as defined in (e) or (f) above wherein the mammal treatedis man.

The following examples illustrate the present invention but should notbe construed as in any way constituting a limitation thereof.

In each of the following examples the APP and BPP, as appropriate, wererespectively the avian pancreatic polypeptide and bovine pancreaticpolypeptide referred to in the foregoing description.

EXAMPLE 1 Injection of APP 5.0 μg/ml

    ______________________________________                                        APP              0.5mg                                                        Dilute Acetic Acid                                                                             sufficient to produce pH 4.0                                 Water for Injections B.P.                                                                      to 100.0ml                                                   ______________________________________                                    

The APP was dissolved in 9/10 of the final volume of Water forInjections adjusted to pH 4.0 with dilute Acetic Acid. The solution wasdiluted to volume with the remaining Water and sterilised by passagethrough a membrane filter, 0.22 μm pore size. The sterile solution wasdistributed aseptically into 1 ml. ampoules which were sealed by fusionof the glass.

An analogous formulation was prepared wherein the APP was replaced byBPP.

EXAMPLE 2 Multidose Injection of APP 50 μg/ml

    ______________________________________                                        APP               5.0mg                                                       Sodium Phosphate Hydrated B.P.                                                                  0.85g                                                       Dilute Phosphoric Acid                                                                          sufficient to produce pH 7.0                                Methyl Hydroxybenzoate                                                                          0.12g                                                       Water for Injections B.P.                                                                       to 100.0ml                                                  ______________________________________                                    

The Methyl Hydroxybenzoate was dissolved in 9/10 of the final volume ofWater at 85° C. The solution was cooled to below 25° C. and the SodiumPhosphate added followed by sufficient Phosphoric Acid to produce pH7.0. The APP was then added and dissolved and the solution diluted tovolume with Water. After sterilisation by passage through a membranefilter, 0.22 μm pore size, the solution was distributed aseptically into10 ml glass vials which were sealed with rubber stoppers secured by analuminum collar.

An analogous formulation was prepared wherein the APP was replaced byBPP.

EXAMPLE 3 Isotonic multidose injection of APP 5 μg/ml

    ______________________________________                                        APP                   0.50mg                                                  Chlorocresol          0.10g                                                   Sodium Chloride       0.86g                                                   Water for Injections B.P.                                                                           to 100.00ml                                             ______________________________________                                    

The Chlorocresol was dissolved in 9/10 of the final volume of Water forInjections at 65° C. After cooling to below 25° C. the Sodium Chloridewas added and dissolved followed by the APP. The injection was dilutedto volume, sterilised and filled into vials as in Example 2.

An analogous formulation was prepared wherein the APP was replaced byBPP.

EXAMPLE 4 Freeze-dried injection of APP 50 μg/vial

    ______________________________________                                        APP              2.5mg                                                        Mannitol         1.0g                                                         Dilute Hydrochloric Acid                                                                       sufficient to produce pH 3.0-4.0                             Water for Injections B.P.                                                                      to 100.0ml                                                   ______________________________________                                    

The APP and Mannitol were dissolved in 9/10 of the final volume of Waterfor Injections and the pH of the solution adjusted to 3.0-4.0 beforediluting to volume. The solution was sterilised by passage through amembrane filter 0.22 μm pore size and distributed aseptically intovials, 2.0 ml/vial. Under aseptic conditions the vials werefreeze-dried. At the end of this process the vials were filled with drysterile Nitrogen and sealed.

The injection is reconstituted before use by the addition of a suitablevolume of sterile diluent such as Water for Injections or PhysiologicalSaline Solution.

An analogous formulation was prepared wherein the APP was replaced byBPP.

EXAMPLE 5

APP (Endocrinology 83, 1323 (1968)) (1 μg) in water (0.1 ml) wasadministered by intraperitoneal injection 3 times a day for 25 days toeach of 5 male mice (age 6 weeks, weight 23 g) of the NZO strain(genetically obese, hyperglycaemic and hyperinsulinaemic). 5 Matchedcontrol animals each received plain water (0.1 ml) by the same route andaccording to the same schedule. The blood glucose and serum insulinlevels were measured at the start and during the course of theexperiment and the mean results for the APP-treated group are shown inTable 1. No change in these levels from the values on day 0, which werethe same as in the treated group, occurred in the control group over thecourse of the experiment.

                  TABLE I                                                         ______________________________________                                                     Blood glucose                                                                             Serum Insulin                                        Day          (mg. %)     (μU/ml)*                                          ______________________________________                                         0           175         189                                                   5           162                                                              10                       99                                                   15           154                                                              20                       47                                                   25           136                                                              ______________________________________                                         *Beef equivalent.                                                        

I claim:
 1. A method of treating a mammal for hyperinsulinaemiacomprising the parenteral administration to the mammal suffering fromhyperinsulinaemia of a non-toxic hyperinsulinaemia treatment effectiveamount of a polypeptide designated Pancreatic Hormone III or apharmaceutically acceptable salt or metal complex of said polypeptide.2. The method of claim 1 wherein said mammal is man.
 3. The method ofclaim 1 wherein said polypeptide is derived from the pancreas of pig, oxor a bird.
 4. The method of claim 1 wherein said polypeptide is derivedfrom the pancreas of an ox.
 5. A method of treating a mammal forhyperinsulinaemia comprising the parenteral administration to the mammalsuffering from hyperinsulinaemia of a non-toxic hyperinsulinaemiatreatment effective amount of a polypeptide wherein said polypeptide hasthe structure:

    ______________________________________                                        Ala.Pro.Leu.Glu.Pro.Gln.Tyr.Pro.Gly.Asp.Asp.Ala.                              1            6            12                                                  Thr.Pro.Glu.Met.Ala.Gln.Tyr.Ala.Ala.Gtu.Leu.                                  13           18           24                                                  Arg.Arg.Tyr.Ile.Asn.Met.Leu.Thr.Arg.Pro.Arg.TyrNH.sub.2                       25           30           36                                                  ______________________________________                                    


6. The method of claim 1 wherein the polypeptide is administered as thefree polypeptide.
 7. The method of claim 1 wherein the polypeptide isadministered as an aqueous solution.
 8. The method of claim 1 whereinthe polypeptide is administered in an amount of from 0.01 to 100.0microgram per kilogram body weight of the animal to be treated.
 9. Themethod of claim 1 wherein said polypeptide is administeredsubcutaneously.